Fig. 3: PREP1 depletion induces actin stress fiber formation. | Communications Biology

Fig. 3: PREP1 depletion induces actin stress fiber formation.

From: The transcription factor PREP1(PKNOX1) regulates nuclear stiffness, the expression of LINC complex proteins and mechanotransduction

Fig. 3

a Confocal images of DAPI (nucleus, blue) staining in control (siLUC) and SUN2 depleted (siSUN2) U2OS cells. Scale bar is 40 µm. Immunoblot shows the knockdown efficiency of SUN2. The bar graph shows percent nuclear deformation in siLUC and siSUN2 cells. N = 576 cells in siLUC and 623 cells in siSUN2. b Confocal images of DAPI staining showing nuclear deformation in control (siLUC), SUN1 depleted (siSUN1), PREP1 depleted (siPREP1) or PREP1 and SUN1 depleted (siPREP1 + siSUN1) U2OS cells, as indicated. Scale bar is 20 µm. White lines in the blot image indicate where an in-between lane was spliced out. The bar graph shows percent nuclear deformation in siLUC, siPREP1 and siPREP1 + siSUN1 conditions. N = 737 cells for siLUC, 670 cells for siPREP1, 551 cells for siSUN1 and 771 cells for siPREP1 + siSUN1. c Representative confocal images of control (siLUC) and PREP1 depleted (siPREP1) U2OS cells showing F-actin (Phalloidin, green) and nucleus (DAPI, blue). Scale bar is 20 µm. d Maximum projection of apical (upper panel showing perinuclear actin stress fibers) or basal (lower panel showing ventral actin stress fibers) confocal stacks of control (siLUC) and PREP1 depleted (siPREP1) HeLa and U2OS cells. Images show F-actin (Phalloidin in green) and nucleus (DAPI in red). Scale bar is 20 µm. e Maximum projection of confocal images of F-actin (Phalloidin, green) and nucleus (DAPI, red) in control (siLUC) and PREP1 depleted (siPREP) U2OS cells grown in fibronectin-coated micropatterns. Right panel shows orthogonal view of the same cells. Scale bar is 20 µm. Bar graph shows the percentage of cells with perinuclear actin cap in siLUC and siPREP1 conditions. N = 33 cells. p = 0.0156. f Maximum projection of confocal z stacks showing nuclear envelope (LAMIN A/C, red) and F-actin (Phalloidin, green) of blebbistatin treated or vehicle (+DMSO) of control (siLUC) and PREP1 depleted (siPREP1) U2OS cells. Scale bar is 40 µm. The bar graph shows quantification of severe nuclear deformation in siLUC and siPREP1 U2OS cells upon treatment with 40 µM Blebbistatin for 20 min. Error bars represent mean ± SE of three independent experiments. N = 898 (siLUC DMSO), 878 (siLUC Blebbistatin), 1067 (siPREP1 DMSO) and 890 cells (siPREP1 Blebbistatin). p = 0.0473.

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